Transcript Ion Channels and Electrical Activity

Ion Channels and Electrical
Activity
The Cell Membrane is Selective
• Criteria for passage through the phospholipid
bilayer:
1. Hydrophobic
2. Net zero charge
3. Nonpolar
4. Size is also a consideration
• Chemicals that will NOT pass through the
phospholipid bilayer:
1. Hydrophillic
2. Charged, ionic
3. Polar
4. Size is also a consideration
• But, using the preceding criteria, many
substances vital to cellular function (e.g., ions)
and survival (e.g., glucose) will not gain entry
into the cell!
• So, nature has come up with channels, which
selectively allow certain substances to gain
entry into the cell, even though they do not
meet the criteria on the preceding slide.
Channels are Vital
Without channels it is energetically unfavorable to
move ions across a membrane –
1. the phospholipid bilayer is ~6-8 nm thick.
2. the hydrophilic head of the phospholipid
molecule projects toward the cytoplasm or the
extracellular fluid.
3. the hydrophobic tails of the phospholipid
molecules project toward each other.
For the Cation to Move Through the
Phospholipid Bilayer…,
1) It must lose its waters of hydration so that it is not so huge
and charged; requires energy to break attractive forces
between the ion and the waters.
2) Energy is also required to move a charged highly
hydrophilic particle into the highly hydrophobic area of
the lipid bilayer that contains the “tails” of the
phospholipid molecules.
3) Based on thermodynamic calculations, so much energy
would be required for this process that it would never
occur.
Channels are thermodynamically similar to Enzymes in that the
former lower the Activation Energy required to move ions across
the Membrane
Transition State
The rate of
the reaction
is determined
by the energy
of activation,
the energy
input
required to
produce the
transition
state.
The uncatalyzed
reaction requires a higher
activation energy than the
catalyzed one does. So, the
latter runs more quickly.
A+B
Reactants
(substrates)
AB
transition
state
C+D
products
There is no difference in free
energy (ΔG) between
uncatalyzed and catalyzed
reactions. The ΔG is the
thermodynamic driving force
for the reaction and determines
the direction of the reaction.
Ion Flow Across the Membrane
• A Chemical can move across the membrane
through one of two ways:
1. Movement through the phospholipid bilayer.
2. Movement through a H2O-filled protein
channel.
Channels are thermodynamically similar to Enzymes in that the
former lower the Activation Energy required to move ions across
the Membrane
The rate of
diffusion is
determined
by the
“energy of
activation”,
the energy
input
required to
produce the
“transition
state”.
(Remove
H2Os of
hydration
and/or move
into hydrophobic
environment.)
Movement through a channel
only requires shedding of
waters of hydration (energy
input would be infinite to
move through the bilayer).
So. Diffusion occurs more
quickly.
ΔG determines this!!
Transition State
Change in free energy is
the thermodynamic
driving force for diffusion
and determines the
direction of ion movement
A+B
Reactants
(substrates)
AB
transition
state
C+D
products
What do we know about the structure of
gated ion channels?
A. Biochemical Information –
1.
2.
3.
4.
MWs range from 25-250 kDal.
They are integral membrane glycoproteins.
They usually consist of 2 or more subunits.
The genes that code for the proteins have been isolated,
cloned and sequenced. These sequences have been
grouped into 6-7 protein families.
5. The primary (amino acid) sequences of these channels
is known.
Use of the Hydrophobicity Plots
1) Propose 3-D structures of the channels
2) Propose functions for specific regions of the
channel proteins
Amino Acid Sequence Enables Ion Channel
Structure Determination
The Voltage Sensor of Ion Channels
• Kv channel: Voltage sensor: S4 – alternating arg and lys
residues (+).
• P(O) determines the overall channel activity.
• P(O) increases as the transmembrane ψ is depolarized.
• P(O) decreases  zero at hyperpolarizing potential.
• Methods used to study:
- Subst cys accessibility method (SCAM) – subst cys at a
specific location in the S4  conformational change.
- FRET – shown that S4 undergoes ~180° rotation upon
depolarization.
Voltage-Gated Na Channels
• H5 loops form part of the ion-conducting pore.
• A specific glu residue within these structures
forms the binding sites for tetrodotoxin and
saxitoxin.
• These negatively charged residues also impart ion
selectivity.
• Na channels inactivate within 1-2 msec 
determines AP duration.
• Ile-Phe-Met residues in the 3rd cytoplasmic loop
(3rd-4th domain) cause the fast inactivation.
Na2+
Na2+
Out
α
α
In
S
S
• Brain Na channel = 1 pore-forming α subunit + 2
auxillary β subunits.
• The β1 subunit responsible for fast inactivation.
• The β1 and β3 isoforms may regulate Na channel
targeting to the Nodes of Ranvier.
• Heart and skeletal muscle express Nav1.4 and
Nav1.5 channels, respectively, where contribute
to saltatory conduction of APs.
• Na channels are held in place by ankyrin G
complexing with NCAMs and ECM components
(e.g., tenascins, phosphacans).
Voltage-Gated K Channels
• Topology and structure of K channel similar to
that of Na channel.
• Most diverse class of ion channels.
• May exist as homomers or as heteropolymers,
which may exist as, e.g., A-B-A-B or A-A-B-B.
Xenopus oocytes as a Heterologous Expression
system for Studying Cloned Ion Channels
• Current-voltage relationship of Kv1.1 currents.
• Plot the normalized peak tail currents
recorded at -50 mV, as a function of the prepulse potentials.
• Shows the fit with the Boltzmann function:
I = 1/[1 + e-(V-V1/2)/k] from which the halfmaximal activation voltage of the channel V1/2
and the steepness of its voltage-dependence
(slope factor k) are calculated.
Inactivation Mechanisms of Kv Channels
• The inactivation of delayed-rectifier K channels
controls neuronal firing properties and their
responses to input stimuli.
• 2 Principle types of inactivation: N- and C-type.
• Ball-and –chain mechanism of pore occlusion.
- Occluding ball = 1st 20 N-terminal aas of Shaker
channels.
- 4 inactivating particles have been found,
although only 1 is enough to occlude the pore.
 very effective occluding mechanism  faster
inactivation than those with only 1 particle
(next slide):
Schematic Diagram of the Molecular
Mechanism of N-type Inactivation
• Based on x-ray crystallography (next slide).
• Upon membrane depolarization, the intracellular gates
open and the positively charged inactivation particle
blocks the channel by entering the central cavity
through 1 of the 4 windows formed by the T1 domains
and the T1-S1 linkers.
• 4 ‘balls and chain’ are provided by the corresponding
auxiliary subunits that are anchored to the T1 domains;
however, only 3 are visible in this figure (both the Kv
and the β subunits are not shown for clarity).
• The Kvβ subunits bind NADP+
(currently not known why)
K+
K+
α1
Cytoplasm
α1
β
Inactivation
Particle
Tether
• Some Kv channels inactivate slowly (C-type
and P-types).
• During intense neural activity, the C-type
inactivation of Kv channels can accumulate,
modifying both the firing rate and the shape
of the AP.
• Involves a conformational Δ of the
extracellular mouth of the pore and a
constriction of the selectivity filter.
Selectivity Filter
• Many channels are selective for only 1 or 2
different chemicals (ions, sugars, etc.).
• The K+ channel has such a filter, which is a
narrow region towards the extracellular surface
of the membrane.
• Two K+ ions can occupy the selectivity filter
simultaneously, with a third in a H2O-filled
cavity deeper in the pore.
Proposed Mechanisms for Channel Ion
Selectivity
Non-specific cation channel, i.e.
little selectivity other than for
cations
Ach receptor
channel - 6.5 A in
diameter
10-20 X more
Na+ than K+
100 X more K+
than Na+
Voltagegated Na+
channel 4 A in
diameter
Voltagegated K+
channel –
3.3 A in
diameter
Proposed Mechanisms for Channel Ion
Selectivity by Channels: Ionic size
Non-specific cation channel, i.e.
little selectivity other than for
cations
Ach receptor
channel - 6.5
A in diameter
If ionic size explains
channel selectivity, why
is the K+ channel so
selective for K+ since
Na+ is smaller?
10-20 X more
Na+ than K+
Voltagegated Na+
channel - 4 A
in diameter
Nonhydrated K+
ion = 2.7 A
in diameter
100 X more K+
than Na+
Voltage-gated
K+ channel –
3.3 A in
diameter
Nonhydrated
Na+ ion =
1.9 A in
diameter
Proposed Mechanisms for Ion Selectivity by
Channels: Ionic size
Non-specific cation channel, i.e.
little selectivity other than for
cations
Ach receptor
channel - 6.5
A in diameter
Modified Model =
perhaps channels select
based on hydrated ionic
radius?
10-20 X more
Na+ than K+
Voltagegated Na+
channel - 4 A
in diameter
100 X more K+
than Na+
Voltage-gated
K+ channel –
3.3 A in
diameter
Hydrated
Na+ ion =
3.3-4 A in
diameter
Hydrated K+
ion = 3.3 A
in diameter
(K+ is larger, has a lower charge
density and so attracts fewer waters
of hydration.)
Proposed Mechanisms for Ion Selectivity by
Channels: Ionic size
The modified model explains K+ channel
selectivity, i.e. the hydrated K+ just fits into the
channel and the hydrated Na+ is too big to fit.
However, how do we explain the +/- sodium
channel selectivity?
A selectivity filter exists inside the channel
Proposed Mechanisms for Ion Selectivity by
Channels: Ionic size
Sodium recognition site =
selectivity filter
Na+
Na+
How might it work? Similar to
enzymes, but much faster?
Evidence for a Selectivity Filter
If channels are simple resistors, than movement
through an open channel should be a function
of the concentration gradient for the ion across
the membrane
Rate of ion movement = a x [ion]I/[ion]o
(current flow = diffusion)
Linear relationship
with slope = a
Evidence for a Selectivity Filter
Observed data for
Na+ channel
Unitary current
(pa) = recordings
from single
channels
Expected data
External [Na+] mM
Evidence for a Selectivity Filter
Data for voltage-gated Na+ channel do not fit the model
of a channel as a simple resistor in the membrane.
Instead, the current flow through the Na+ channel
plateaus or “saturates” at high [Na+]. This relationship
looks like what happens to an enzyme at high
[substrate]. Perhaps some channels select ions
based on the same biochemical mechanisms used
by enzymes to select their substrates?
In the end, the final determinations of channel gating
mechanisms and ion selectivities will come from Xray crystallography of the purified channels.
Inward Rectifying K Channels and Cell
Excitability
• 2 transmembrane domains separated by a K+
pore sequence and can assemble as both
homotetramers and heterotetramers.
• Acts as a diode: Iinward > Ψ< EK. But at more
positive Ψs, Ioutward is inhibited and the Ψ is
therefore, free to change.
• Rectifying nature of the conductance is because
of a voltage-dependent block of the intracellular
side of the pore by cytoplasmic polyamines and
Mg2+ ions.
K Selectivity of Kv Channels
(next slide)
• The H5 loop contributes to the ion-conducting
pore.
• Site-directed mutagenesis of the H5 loop: GYG.
• GYG acts as the K+ selectivity filter, which is lined
with carbonyl O atoms.
• X-ray crystallography: 3.2A resolution; central
cavity diameter = 10A, channel length = 12A.
• The pore contains 2 K+ ions, 7.5A apart.
• These dimensions optimal for rapid conduction
and selectivity.
KcsA channel
expressed in
S lividans,
highly homologous
to the mammalian
Kir channel.
• Inhibitory neurotransmitters exert their inhibitory
actions by activating G-protein coupled inward
rectifiers.
• These channels belong to the Kir3.x family.
• Regulate excitability in brain and heart.
• The dissociated βγ subunits stimulate
heteromeric Kir3.1/Kir3.4 channel activity by
physically interacting with their intracellular
termini.
• Recall: activation of these channels results in an
efflux of K+ ions that causes membrane
hyperpolarization and cell inhibition.
• KATP channels – another important group of
inward rectifier K channels.
• Insulin secretion from pancreatic β-cells is
mediated by the closure of these channels caused
by increased levels of ATPcytoplasm.
• KATP channel = 4 Kir6.2 subunits + 4 sulfonylurea
receptors (sensitivity to drugs).
• ATP inhibits channel opening by interacting with
the Kir6.2 subunits.
• Expressed in brain and heart, where they couple
metabolic state of the cell to electrical activity.
Voltage-Gated Ca Channels
• Activity initiated by depolarizing stimuli.
• Ca2+ influx down a steep electrical and
chemical gradient.
• Can be depolarized by weak or strong stimuli:
called low-threshold or high-threshold Ca2+
channels.
• More detailed electrophysiological studies
revealed several channel types: L-, P-, Q-, N-,
R-, and T-types
VG Ca Channel Types and Voltage-Dependency
•
•
•
•
Cav1.1
Cav1.2
Cav1.3
Cav1.4
L-Type
High-Threshold (>-10 mV)
• Cav2.1
• Cav2.2
• Cav2.3
P/Q-Type
N-Type
R-Type
High-Threshold (>-20 mV)
• Cav3.1
mV)
• Cav3.2
• Cav3.3
T-Type
Low-Threshold (>-70
L-Type
• High To (> -10 mV).
• Long-lasting openings, slow inactivation.
• In muscle, are voltage sensors for excitationcontraction coupling.
• In heart, β-adrenergic receptors stimulate
activity of Cav1.2 channels to enhance cardiac
contractility and excitability (pace maker
cells).
• Cav1.2 and Cav1.3 widely expressed and
involved in hippocampal-dependent plasticity
via NMDA receptors
P/Q-Type:
• Cav2.1 (P/Q type) abundantly and widely
expressed throughout the CNS, regulate fast
synaptic transmisssion neuron survival,
excitability, gene expression, and plasticity.
• Located at the NMJ and at most presynaptic
terminals in the cerebellum.
• SNARE protein complex.
• Undergos alt. splicing of the α1A gene.
• KO mice for P/Q type exhibit ataxia, dystonia,
and Purkinje cell death.
N-Type:
• Expressed in sympathetic nervous system.
• Voltage dependence modulated by
neurotransmitters via GPCRs.
• Activation of the Gβγ subunits inhibits Cav2.2
channels by decrease the mobility of the
voltage sensor.
• Thus, the currents show slower time courses
of activation and decreased voltagedependence.
T-Type:
• Cav3.x – low threshold, characterized by
transient kinetics, small single channel
conductance and fast inactivation.
• Various isoforms (splice variants) display
various kinetics of inactivation.
• Kinetics determined by the N-terminal
domains of the β subunits.
Ca2+
α2
γ
δ
S
S
Out
α1
In
β